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1、VIROLOGY75,155-165(1976)PurificationandBiophysicalPropertiesofHumanCoronavirus229EJOHNC.HIERHOLZERRespiratoryVirologyBranch,VirologyDivision,BureauofLaboratories,CenterforDiseaseControl,PublicHealthService,DepartmentofHealth,EducationandWelfare,Atlanta,Georgia30333Accepted
2、July9,1976Coronavirus2293wasgrowntohightitersindiploidfibroblastcellsundermediumcontainingtwicethenormalconcentrationsofaminoacidsandvitamins.Growthcurvesshowedmaximumvirusproductionatmultiplicitiesofinfectionof0.1and1;maximumtitersofintracellularvirusoccurredat22-24hrando
3、fextracellularvirusat26hrpostadsorption.Tubeinfectivitytitersrangedfrom109.-105TCID,,/mlandplaquetitersfrom10.-lOO-PFU/mlatthetimeofpeakvirusproduction,whennocytopathol-ogywasevident.Virustiterdroppedrapidlybetween26and56hr,coincidentwithincreasingcytopathology.Asingleprec
4、ipitinbandwasobservedinimmunodiffusionandimmunoelectrophoresisbetweenconcentratedviruspreparationsandantiserumtopurified2293.Neuraminidaseandhemagglutininassayswerenegative.Viruswaspurifiedbytwoprocedures:adsorptiontoandelutionfromhuman0erythrocytesandCaHPO,gelfollowedbyeq
5、uilibriumsucrosegradientcentrifugation,andPEGprecipi-tationfollowedbyequilibriumglycerol/tartrategradientsandratezonalsucroseorglycerolitartrategradients.Finallotsofpurifiedviruscontaining<0.02%ofthecrudetissuecultureproteinshadabsorptionmaximaat256nmandminimaat241.2nmanda
6、meanextinctioncoefficientofE:$=54.3at256nm.ThefullycorrectedsedimentationcoefficientfortheintactvirionwasS&,h=381S.PAGEbydifferenttechniquesrevealedsevenpolypeptidesofmeanapparentmolecularweightsbetween16,900and196,100.Sixcontainedcarbohydrateandonecontainedlipid.Electroph
7、erogramsof3H-andC-labeledviruswereidenticaltothoseofstainedgels.Twoglycoproteinsconstituting25%ofthevirionproteinwereidentifiedbybromelindigestionasthespikeproteins.Thedensityinsucroseandinpotassiumtartratewas1.18g/mlforthevirionand1.15g/mlforthedespikedparticle.INTRODUCTI
8、ONvirus(TGE)ofpiglets,acuteenteritisco-ronavirus1-71ofneonataldogs(KeenanetThecoronavirus
